Sensitive non-radioactive determination of aminotransferase stereospecificity for C-4' hydrogen transfer on the coenzyme |
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Authors: | Jomrit Juntratip Summpunn Pijug Meevootisom Vithaya Wiyakrutta Suthep |
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Institution: | aDepartment of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand;bDepartment of Microbiology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand;cCenter of Excellence for Agricultural Biotechnology: (AG-BIO/PERDO-CHE), Bangkok, Thailand |
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Abstract: | A sensitive non-radioactive method for determination of the stereospecificity of the C-4′ hydrogen transfer on the coenzymes (pyridoxal phosphate, PLP; and pyridoxamine phosphate, PMP) of aminotransferases has been developed. Aminotransferase of unknown stereospecificity in its PLP form was incubated in 2H2O with a substrate amino acid resulted in PMP labeled with deuterium at C-4′ in the pro-S or pro-R configuration according to the stereospecificity of the aminotransferase tested. The 4′-2H]PMP was isolated from the enzyme protein and divided into two portions. The first portion was incubated in aqueous buffer with apo-aspartate aminotransferase (a reference si-face specific enzyme), and the other was incubated with apo-branched-chain amino acid aminotransferase (a reference re-face specific enzyme) in the presence of a substrate 2-oxo acid. The 2H at C-4′ is retained with the PLP if the aminotransferase in question transfers C-4′ hydrogen on the opposite face of the coenzyme compared with the reference aminotransferase, but the 2H is removed if the test and reference aminotransferases catalyze hydrogen transfer on the same face. PLP formed in the final reactions was analyzed by LC–MS/MS for the presence or absence of 2H. The method was highly sensitive that for the aminotransferase with ca. 50 kDa subunit molecular weight, only 2 mg of the enzyme was sufficient for the whole test. With this method, the use of radioactive substances could be avoided without compromising the sensitivity of the assay. |
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Keywords: | Aminotransferase Hydrogen transfer LC&ndash MS/MS Non-radioactive Pyridoxal phosphate Stereospecificity |
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